TITLE: Multifunctional PSCA Antibody Fragments for PET and Optical Prostate Cancer Imaging

Transcription

1 AWARD NUMBER: W81XWH TITLE: Multifunctional PSCA Antibody Fragments for PET and Optical Prostate Cancer Imaging PRINCIPAL INVESTIGATOR: Anna M. Wu CONTRACTING ORGANIZATION: University of California, Los Angeles Los Angeles, CA REPORT DATE: October 2016 TYPE OF REPORT: Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland DISTRIBUTION STATEMENT: Approved for Public Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation.

2 REPORT DOCUMENTATION PAGE Form Approved OMB No Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports ( ), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE October REPORT TYPE Annual 3. DATES COVERED 30 Sep Sep TITLE AND SUBTITLE 5a. CONTRACT NUMBER Multifunctional PSCA Antibody Fragments for PET and Optical Prostate Cancer Imaging 5b. GRANT NUMBER W81XWH c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Anna M. Wu awu@mednet.ucla.edu 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) University of California, Los Angeles Kinross Avenue, Suite 211 Los Angeles, CA d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 8. PERFORMING ORGANIZATION REPORT NUMBER 9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland SPONSOR/MONITOR S REPORT NUMBER(S) 12. DISTRIBUTION / AVAILABILITY STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The goals of this initial year of funding were to develop engineered antibody fragments (cys-diabodies and cys-minibodies) suitable for labeling with positron-emitting radionuclides and fluorescent dyes for multimodal imaging of prostate cancer. A novel cys-minibody recognizing PSCA (prostate stem cell antigen) has been produced. Conjugation conditions have been developed for radiolabeling with either and I-124 and Zr-89 via lysine (random) or cysteine (site-specific) residues of the proteins. Targeting, imaging, and biodistribution studies in mouse models of prostate cancer confirm high activity in PSCA-positive tumors with excellent visualization by PET imaging. Recently, dual-labeling (with radionuclide and fluorescent dye) of the cysminibody has been developed and evaluated in mouse models. 15. SUBJECT TERMS Prostate cancer, imaging, antibody fragment, Positron emission tomography, fluorescence imaging, PSCA 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC a. REPORT U b. ABSTRACT U c. THIS PAGE U UU 10 19b. TELEPHONE NUMBER (include area code) Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std. Z39.18

3 Table of Contents Page 1. Introduction Keywords Accomplishments Impact Changes/Problems Products Participants & Other Collaborating Organizations Special Reporting Requirements Appendices 10

4 1. INTRODUCTION Imaging remains a major unmet need in the management of prostate cancer. We are developing imaging probes based on engineered antibodies that recognize PSCA (prostate stem cell antigen), a cell surface protein highly expressed in prostate cancer. These engineered antibody fragments (cysminibodies and cys-diabodies) can be labeled with radioisotopes for non-invasive PET imaging for use at multiple points in the prostate cancer treatment continuum, including staging at diagnosis, monitoring treatment, and re-staging at various points during management. Engineered fragments can also be labeled with fluorescent dyes for visual guidance in an intraoperative setting to ensure complete resection with negative margins. In this project, dually-labeled PSCA imaging agents are being developed that can be used for pre- and intra-operative detection of prostate cancer. 2. KEYWORDS Prostate cancer, imaging, antibody fragment, positron emission tomography, fluorescence imaging, PSCA 3. ACCOMPLISHMENTS What were the major goals of the project? Specific Aim 1. Develop universal optimized cys-diabody and cys-minibody fragments against PSCA for PET imaging of prostate and pancreatic cancer. Subtasks: Major Task 1. Develop and evaluate cys-diabody and cys-minibody fragments Major Task 2. Design, optimize and test multifunctional, F-18, and alternatively labeled fragments Major Task 3. New technologies: alternative site-specific labeling methods, use of click chemistry Specific Aim 2. Evaluate the ability of lead PSCA fragments to imaging prostate cancer in disease progression in xenograft and genetically engineered models of prostate cancer Subtasks: Major Task 4. Image bone and lymph node in xenograft models Major Task 5. Image transgenic mouse models Major Task 6. Development and evaluation of singly labeled and optimized optical probes for surgery Major Task 7. Development of dual labeled probes for PET and optical imaging. For Dr. Wu (Partnering PI) during Year 1 of the project, the subtasks were: Major Task 1 Subtask 1. Produce and purify A2 cys-diabody and A2-cys-minibody in mg quantities (months 1-6) Completed. Subtask 2. Optimize radiolabeling conditions with I-124 (tyrosine) and Zr-89 (DFO conjugation to lysine and cysteine residues). Confirm retention of binding by QCM and cell binding. (months 3-9) Completed. 4

5 Subtask 3. Conduct micropet imaging and biodistribution in subcutaneous models using I- 124 and/or Zr-89; provide PET tracers to Aim 2. (months 6-18) Completed and ongoing. What was accomplished under these goals? Year 1 has focused on the development, production, and optimization of the PET imaging agents. Two closely-related sequence variants, A2 and A11 have been evaluated. Of note, a novel A11 cysminibody has been designed, produced, and purified, and this has been designated the lead candidate for singly and dually-labeled imaging agents. The protein has been radiolabeled with Zr-89 (sitespecific) (Figure 1) and I-124 (Figure 2) for PET imaging studies. Figure 1. Imaging and biodistribution of A11 cysminibody site-specifically conjugated using maleimide DFO and radiolabeled with 89Zr. Left, diagram of cys-minibody with C-terminal thiols and radionuclide. Center, immunopet imaging of PSCA(-) and PSCA (+) tumors at 24 and 44 h postinjection showing antigen-driven localization and renal clearance. Right, biodistribution. Importantly, significant progress has been made ahead of schedule on production and evaluation of a dually-labeled PET/optical probe, as detailed in the abstract below, which was presented at the Antibody Technology Resource Center Symposium at UC San Francisco, October Dual-modality immuno-pet/fluorescence imaging of prostate cancer using anti-psca A11 cys-minibody Wen-Ting Tsai 1, Kirstin Zettlitz 1, Richard Tavaré 1, Felix Salazar 1, Robert Reiter 2, and Anna Wu 1 1 Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA 2 Department of Urology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Prostate cancer can benefit from non-invasive and more accurate diagnosis, as well as improved visualization during surgery. Immuno-PET can provide information on extent and location of the disease, while fluorescent image-guided surgery can distinguish cancerous tissue from healthy surrounding tissues for clean resection. Prostate Stem Cell Antigen (PSCA) is upregulated in the majority of prostate cancers and metastases and is therefore a promising target for imaging (Knowles J Nucl Med 55:429, 2014). Engineered antibody fragments, such as the minibody, exhibit ideal immuno-pet imaging characteristics due to fast blood clearance for high target-to-background images at short imaging times post-injection (Wu Methods 65:139, 2014). A dual-labeled minibody probe can reveal the current PSCA-expressing tumor burden by PET, while also identifying margins of malignancy by near-infrared fluorescence. The humanized anti-psca A11 minibody (A11 Mb) was previously affinity matured by yeast scfv display (Lepin Eur. J. Nucl. Med. 37:1529, 2010), then engineered with a C-terminal cys-tag (A11 cmb) that can be site-specifically labeled by thiol-chemistry. 124 I-A11 cmb, 124 I-Cy5.5-A11 cmb, 89 Zr-DFO-A11 cmb, and 89 Zr-DFO-A11 cmb-cy5.5 were used to image 22Rv1 tumors expressing PSCA. For dual-modality imaging, A11 cmb was site-specifically conjugated with Cy5.5-maleimide and radiolabeled with 124 I or 89 Zr. PET imaging with 124 I-Cy5.5-A11 cmb in nude mice bearing 5

6 subcutaneous PSCA-positive and negative tumors resulted in a positive-to-negative tumor ratio of 13:1 at 22 hours post-injection, comparable to the 8:1 ratio when imaged with 124 I-A11 cmb. The PSCA-positive tumors were subsequently visualized by fluorescence in situ and ex vivo (see Figure 1). In order to radiolabel with 89 Zr, a metal chelator desferrioxamine (DFO) was conjugated to A11 cmb by maleimide chemistry, or SCN-DFO was labeled to random lysines. 89 Zr-DFO-A11 cmb demonstrated specific tumor targeting in subcutaneous PSCA-positive tumors. In an orthotopic model, imaging with 89 Zr-DFO-A11 cmb-cy5.5 resulted in a 3:1 tumor-to-blood ratio, and fluorescence clearly distinguished prostate tumor from adjacent tissues. In conclusion, the novel A11 cmb has been successfully used for visualization of tumor burden by immuno-pet and fluorescence imaging, which has the potential for clinical translation. 124 I-A11-cys minibody 124 I-A11-Cy5.5-cys 4 hr minibody 22 hr Figure 2. Dually-labeled A11 anti-psca cys-minibody. A11 cys-minibody was reduced using TCEP and site-specifically conjugated to maleimide Cy5.5 fluorescent dye. Non-conjugated and dye-conjugated cys-minibodies were then radiolabeled with I-124 for PET imaging. Mice bearing 22rv1 (right shoulder) and 22rv1-PSCA (left shoulder) were injected with singly- or dually-labeled A11 PSCA cys-minibodies. Serial PET imaging at 4 and 22 hrs show excellent localization of both probes to PSCA+ tumors by 22 h post injection. Mice were euthanized, and subject to fluorescent imaging following removal of skin (top right); isolated tumors were subsequently removed and also imaged optically (bottom right). Ongoing activities include collaborative studies with Co-investigator Dr. Jennifer Murphy and her laboratory, developing site-specific F-18 radiolabeling approaches (Major Task 2, Subtask 2). We have also embarked on the development of multifunctional linkers that will carry both the PET and optical tags (Major Task 2, subtask 5). Finally, work continues on development of click chemistry approaches for rapid, efficient radiolabeling (Major Task 3, subtask 2). We expect to report on progress on these tasks next year. We also attempted to develop and implement a site-specific conjugation approach based on introduction of specific mannosylation sites into the engineered proteins. (Major Task 2, Subtask 4 and Major Task 3, Subtask 1). Thus far, efficiency of mannosylation has been too low to be useful. We will continue to try to develop this approach but it may not be feasible. Regardless, as our overall strategy included parallel approaches to achieve our goals, we will still be able to achieve our 6

7 overall objectives, as demonstrated by our success with the dually-labeled cys-minibodies, described above. What opportunities for training and professional development has the project provided? Nothing to report. How were results disseminated to communities of interest? Nothing to report What do you plan to do during the next reporting period to accomplish the goals? During the next year we will be able to transition our lead dually-labeled PET/optical probes to more biologically relevant models of prostate cancer including bone and lymph node metastasis models as well as genetically engineered mouse models, in close collaboration with the overall PI, Dr. Robert Reiter. We will also begin to apply these in intraoperative models of prostate cancer, again in with Dr. Reiter. Finally, we will focus on the dual F-18/optical probes (which will be based on the smaller A2 cys-diabody because its in vivo kinetics are better matched to the physical half-life of F-18). 4. IMPACT What was the impact on the development of the principle discipline of the project? Nothing to report. What was the impact on other disciplines? Nothing to report. What was the impact on technology transfer? Nothing to report. What was the impact on society beyond science and technology? Nothing to report. After only one year of funded research it is too early for our findings to have significant impact. 5. CHANGES/PROBLEMS Nothing to report. There are no significant changes to the objectives, scope, and approaches of the project. 6. PRODUCTS Publications, conference papers, and presentations Abstracts 7

8 Tsai, Wen-ting, Tavaré, R., Zettlitz, K.A., Salazar, F.B., Knowles, S., Reiter, R., and Wu, A.M. (2015). Dual modality immunopet/fluorescence imaging of prostate cancer. World Molecular Imaging Congress, Honolulu, HI. Tsai, W.-T., Tavaré, R., Zettlitz, K.A., Salazar, F.B., Reiter, R.E., and Wu, A.M. (2015) Dualmodality immunopet/fluorescence imaging of prostate cancer using anti-psca cys-minibody. Antibody Engineering and Therapeutics 2015, San Diego, CA. Tsai, W.-T., Zettlitz, K., Tavaré, R., Salazar, F., Reiter, R., and Wu, A. Dual-modality immune- PET/fluorescence imaging of prostate cancer using anti-psca A11 cys-minibody. (2016) Antibody Technology Resource Center Symposium, San Francisco, CA. 7. PARTICIPANTS AND OTHER COLLABORATING ORGANIZATIONS What individuals have worked on the project? Name: Anna Wu Project Role: Partnering Principal Investigator Researcher Identifier (e.g. ORCID ID): Nearest person month worked: 1 Dr. Wu oversaw all aspects of work performed and Contribution to Project: accomplished to-date. Funding Support: Name: Jennifer Murphy Project Role: Co-Investigator Researcher Identifier (e.g. ORCID ID): Nearest person month worked: 1 Dr. Murphy had performed work on the design and Contribution to Project: evaluation of the chemical and radiochemical strategies for combined PET/optical labeling. Funding Support: Name: Project Role: Researcher Identifier (e.g. ORCID ID): Amanda Freise Graduate Student 8

9 Nearest person month worked: 2 Contribution to Project: Funding Support: Ms. Freise had performed work on site-specific conjugation, radiolabeling, and imaging of engineered antibody fragments. Name: Project Role: Researcher Identifier (e.g. ORCID ID): Nearest person month worked: 2 Contribution to Project: Funding Support: Christopher Waldmann Postdoctoral Researcher Dr. Waldmann had performed work on developing methods for F-18 labeling of engineered antibody fragments. Has there been a change in the active other support of the PD/PI(s) or senior/key personnel since the last reporting period? Changes in active other support of PI (Dr. Anna Wu) The following funding has ended (reported in original grant submission): NIH R01 CA Antibody-CPG conjugates for the treatment of B cell lymphoma (Timmerman, PI) NIH U54 CA Center for Cancer Nanotechnology Excellence and Translation (Gambhir, PI) NIH R25 CA UCLA Scholars in Oncologic Molecular Imaging (Phelps, PI) NIH R21 AI In vivo imaging of T cells using engineered antibodies and PET (Wu, PI) NIH R21 CA190044) (PQC4) Imaging CD8 T cells in tumor immunotherapy by immunopet (Wu, PI) The following funding has been renewed and/or is active: Renewal funding: R01CA Multifunctional immunopet tracers for pancreatic and prostate cancer (Wu, Reiter, Multi-PIs); time commitment of 1.8 calendar months; renewal project period of 8/1/2016-7/31/2021 New funding: Cornell University (NSF prime) SNM: Scalable Cell-free Protein Manufacturuing via NanoClay-DNA (NanoCD) Microdonuts (Luo, PI); time commitment of 0.6 calendar months; project period of 9/15/2015 8/31/2019 9

10 Changes in active other support of Co-Investigator (Dr. Jennifer Murphy) New funding: R01CA Multifunctional immunopet tracers for pancreatic and prostate cancer (Wu, Reiter, Multi-PIs); time commitment of 0.60 calendar months; project period of 8/1/2016 7/31/2021 What other organizations were involved as partners? Nothing to report. 8. SPECIAL REPORTING REQUIREMENTS Collaborative awards This report covers the activities of the Partner PI, Dr. Anna Wu. 9. APPENDICES None 10